CRISPR Aids New Zika Paper-Based Diagnostic Test

[Jose-Luis Olivares/MIT. Background image by CDC/Cynthia Goldsmith]

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, Keith Pardee, Ph.D., discusses how a low-cost, rapid diagnostic test for the detection of the Zika virus works.

The rapid spread of Zika virus into South America and its link to fetal neurodevelopmental issues have underscored the need for fast, low-cost diagnostics for use in endemic regions. Now, a collaborative team of scientists from Wyss Institute for Biologically Inspired Engineering, Massachusetts Institute of Technology, Boston University, Arizona State University, Cornell University, University of Wisconsin-Madison, Broad Institute, and the University of Toronto have developed a cell-free, paper-based platform that can host synthetic gene networks and help reliable diagnosis of Zika-infected patients.

This new test, which can distinguish Zika from the very similar dengue virus within a few hours, can be stored at room temperature and read with a simple electronic reader, making it practical for widespread use.

"One of the key problems in the field is being able to distinguish what these patients have in areas where these viruses are co-circulating," explained coauthor Lee Gehrke, Ph.D., professor of microbiology and immunobiology at Harvard Medical School.

"We [now] have a system that could be widely distributed and used in the field with low cost and very few resources," added senior study author James Collins, Ph.D., professor of medical engineering and science in MIT's Department of Biological Engineering and Institute for Medical Engineering and Science (IMES).

The findings from this study were published recently in Cell in an article entitled “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.”

Currently, patients are diagnosed by testing for reactive antibodies against Zika in their bloodstream or by looking for pieces of the viral genome in a patient's blood sample using PCR. However, these tests can take days or weeks to yield results. Furthermore, the antibody test cannot discriminate accurately between Zika and dengue.

The researcher team created a cluster of diagnostic measuring devices on a freeze-dried piece of paper the size of a stamp that employs toehold sensors and isothermal RNA amplification—coupled to a CRISPR based module. Activated by the amplified sample, the diagnostic sensors programmed into the paper provide an extremely sensitive, low-cost, programmable assay that provides rapid results.

The new test is based on technology that Dr. Collins and colleagues previously developed to detect the Ebola virus. In October 2014, the researchers demonstrated that they could create synthetic gene networks and embed them on small discs of paper. These gene networks can be programmed to detect a particular genetic sequence, which causes the paper to change color.

The sensors, embedded in the paper discs, can detect 24 different RNA sequences found in the Zika viral genome, which, like that of many viruses, is composed of RNA instead of DNA. When the target RNA sequence is present, it initiates a series of interactions that turns the paper from yellow to purple, which can be visualized by eye. The researchers also developed an electronic reader that makes it easier to quantify the change, especially in cases where the sensor is detecting more than one RNA sequence.

The investigators tested the device with samples taken from monkeys infected with the Zika virus because samples from human patients affected by the current Zika outbreak have been difficult to obtain. Amazingly, they found that in these samples, the device could detect viral RNA concentrations as low as 2 or 3 parts per quadrillion.

“The diagnostic platform developed by our team has provided a high-performing, low-cost tool that can work in remote locations," noted lead study author Keith Pardee, Ph.D., assistant professor at the University of Toronto. "We have developed a workflow that combines molecular tools to provide diagnostics that can be read out on a piece of paper no larger than a postage stamp. We hope that through this work, we have created the template for a tool that can make a positive impact on public health across the globe."

"Our synthetic biology pipeline for rapid sensor design and prototyping has tremendous potential for application for the Zika virus and other public health threats, enabling us to rapidly develop new diagnostics when and where they are needed most," Dr. Pardee added.

The researchers have envisioned that this approach could also be adapted to other viruses that may emerge in the future. Dr. Collins now hopes to team up with other scientists to develop the technology further for diagnosing Zika.

"Here we've done a nice proof-of-principle demonstration, but more work and additional testing would be needed to ensure safety and efficacy before actual deployment," Dr. Collins said. "We're not far off."